Tohoku J. Exp. Med., 2011,Differential 223, 161-176 Gene Expression in OPCs, Oligodendrocytes and Type II Astrocytes 161 Differential Gene Expression in Oligodendrocyte Progenitor Cells, Oligodendrocytes and Type II Astrocytes Jian-Guo Hu,1,2,* Yan-Xia Wang,3,* Jian-Sheng Zhou,2 Chang-Jie Chen,4 Feng-Chao Wang,1 Xing-Wu Li1 and He-Zuo Lü1,2 1Department of Clinical Laboratory Science, The First Affiliated Hospital of Bengbu Medical College, Bengbu, P.R. China 2Anhui Key Laboratory of Tissue Transplantation, Bengbu Medical College, Bengbu, P.R. China 3Department of Neurobiology, Shanghai Jiaotong University School of Medicine, Shanghai, P.R. China 4Department of Laboratory Medicine, Bengbu Medical College, Bengbu, P.R. China Oligodendrocyte precursor cells (OPCs) are bipotential progenitor cells that can differentiate into myelin-forming oligodendrocytes or functionally undetermined type II astrocytes. Transplantation of OPCs is an attractive therapy for demyelinating diseases. However, due to their bipotential differentiation potential, the majority of OPCs differentiate into astrocytes at transplanted sites. It is therefore important to understand the molecular mechanisms that regulate the transition from OPCs to oligodendrocytes or astrocytes. In this study, we isolated OPCs from the spinal cords of rat embryos (16 days old) and induced them to differentiate into oligodendrocytes or type II astrocytes in the absence or presence of 10% fetal bovine serum, respectively. RNAs were extracted from each cell population and hybridized to GeneChip with 28,700 rat genes. Using the criterion of fold change > 4 in the expression level, we identified 83 genes that were up-regulated and 89 genes that were down-regulated in oligodendrocytes, and 92 genes that were up-regulated and 86 that were down-regulated in type II astrocytes compared with OPCs. The up-regulated genes, such as activating transcription factor 3 and myelin basic protein in oligodendrocytes or claudin 11 in type II astrocytes, might contribute to OPC differentiation and represent constitutive components of oligodendrocytes or type II astrocytes. The down-regulated genes in both oligodendrocytes and type II astrocytes, such as transcription factor 19, might be involved in maintaining self-renewal and/or represent the property of OPCs. These results provide new insights into the elucidation of the molecular mechanisms, by which OPCs differentiate to oligodendrocytes or type II astrocytes. Keywords: gene expression; cDNA microarray; oligodendrocyte precursor cells; oligodendrocytes; type II astrocytes Tohoku J. Exp. Med., 2011, 223 (3), 161-176. © 2011 Tohoku University Medical Press Oligodendrocytes (OLs) produce myelin in the central taining medium when cultured in vitro (Raff et al. 1983; nervous system (CNS). They wrap axons of neurons to Louis et al. 1992). Type II astrocytes displayed a typical produce myelin sheaths, provide trophic support and pro- process-bearing morphology and express both A2B5 and tection for neurons and their axons. The persistent defi- glial fibrillary acidic protein (GFAP) and may contribute to ciency in myelin results in the disorders of the CNS, such the structure of nodes of Ranvier (Ffrench-Constant and as multiple sclerosis and spinal cord injury. Oligodendro - Raff 1986). OPC transplantation into the area of demyelin- cytes (OLs) are generated from oligodendrocyte precursor ation may be a feasible approach to repairing CNS disor- cells (OPCs), which arise at multiple locations in the devel- ders (Chari and Blakemore 2002; Keirstead et al. 2005). oping brain and spinal cord and migrate throughout the However, due to their bipotential differentiation potential, CNS (Qi et al. 2002; Kessaris et al. 2006). OPCs express when transplanted into lesion site, the majority of OPCs specific markers, such as a cell-surface ganglioside (A2B5) differentiate into astrocytes (Lu et al. 2010). Therefore, it is that is recognized by A2B5 monoclonal antibody (Raff et an important issue to control the implanted OPCs to differ- al. 1983) and platelet-derived growth factor receptor α entiate into OLs, but not astrocytes. For this purpose, the (PDGFRα), and they are bipotential in vitro (Raff et al. molecular mechanisms that control OPC differentiation 1983; Louis et al. 1992). OPCs can differentiate into OLs should be well understood. in serum-free medium or type II astrocytes in serum-con- Microarray technology provides a powerful tool for Received December 30, 2010; revision accepted for publication January 29, 2011. doi: 10.1620/tjem.223.161 *These authors contributed equally. Correspondence: He-Zuo Lü, Ph.D., Anhui Key Laboratory of Tissue Transplantation, Bengbu Medical College, 287 Chang Huai Road, Bengbu 233004, P.R. China. e-mail: [email protected] 161 162 J.G. Hu et al. large-scale gene expression studies (Hu et al. 2004). In the Chemicon), the markers for OLs, or both A2B5 and glial fibrillary present study, we cultured OPCs isolated from the spinal acidic protein (GFAP; 1:200; Sigma, St. Louis, MO, USA), the cords of embryonic rats at 16 days of gestation and induced marker for type II astrocytes overnight at 4°C. On the second day, them into OLs or type II astrocytes under different culture the cultures were incubated with rhodamine- or fluorescein isothiocy- conditions, respectively. We then employed a cDNA micro- anate (FITC)-conjugated secondary antibody (all from Jackson array to detect differential gene expression in three cell ImmunoResearch Lab, West Grove, PA, USA) for 1 hour at 37°C. After staining, the coverslips were rinsed and mounted with Gel/ types. Mount aqueous mounting media (Biomeda Corp., Foster City, CA, Materials and Methods USA) containing Hoechst 33342, a fluorescent nuclear dye (1 μg/ml; Sigma). The coverslips were examined using an Olympus BX60 Cell Culture microscope. For cell counts, at least five randomly selected fields OPCs were immunopanned from embryonic day 16 (E16) with a total of more than 500 cells were counted. In all experiments, Wistar rat spinal cords using an A2B5 antibody and a protocol modi- primary antibody omission controls were used to confirm the specific- fied from previous studies (Mayer-Proschel et al. 1997; Mujtaba et al. ity of the immunofluorescence labeling. 1999; Cao et al. 2005). Immunopanned OPCs were plated onto Poly- D-lysine (PDL)/fibronectin-coated 10 × 12 cm culture dishes and growth medium was added and changed every other day. The growth RNA Isolation and Hybridization to Affymetrix GeneChips medium contained DMEM/Ham’s F12 (Invitrogen, Carlsbad, CA, OPCs, differentiated OLs and type II astrocytes (5 days) derived USA), 1 × N2 and 1 × B27 supplements (Invitrogen), fibroblast from three rats were used in cDNA microarray detection. Purified growth factor 2 (FGF-2) (20 ng/ml; Sigma, St. Louis, MO), and cells were rinsed twice with PBS and total RNA was extracted using platelet-derived growth factor aa (PDGF-aa) (10 ng/ml; Chemicon, SV total RNA isolation system (Promega, Madison, USA) according Temecula, CA). After 5~7 days, the cells were passaged. In all cases, to the manufacturer’s instruction. RNA was treated with DNase I an aliquot of cells was analyzed the following day to determine the (Promega) to eliminate genomic DNA contamination. The integrity efficiency of immunopanning. Only those preparations in which and purification of RNA samples were monitored by formaldehyde/ >95% of the bound cells expressed A2B5 and at passage 2 (P2) were agarose gel electrophoresis. The concentration of RNA was deter- used. OLs and type II astrocytes were induced from the OPCs in the mined by repeated OD measurements of aliquots at wavelength of presence of thyroid hormone [tri-iodothyronine (T3)] or serum after 260 nm. withdrawal of bFGF and PDGF-AA according to the method The microarray hybridization analysis was performed by described previously (Fu et al. 2007; Lu et al. 2008). Briefly, for OL Biochip Co., Ltd. (Shanghai, China) according to the manufacturer’s differentiation, PDGF and bFGF were withdrawn from the OPC- protocol (Affymetrix, Santa Clara, CA, USA). Biotinylated cRNA TM medium, T3 was added (30 μM). The OPCs were allowed to differ- samples were prepared according to the standard Enzo Bioarray entiate for 5 days. Antibodies against O1 (a marker of pre-myelinat- protocol (Enzo Life Sciences, Farmingdale, NY, USA). In brief, total ing OLs), receptor interaction protein (Rip) and myelin basic protein RNA (1 μg) from each sample was converted to double-stranded TM (MBP) were used to identify OLs. For type 2-astrocytes differentia- cDNA with the Bioarray Single-Round RNA Amplification and tion, OPCs were cultured in basal-OPC-medium supplemented with Labeling Kit (Enzo Life Sciences). After second-strand synthesis, the 10% fetal bovine serum (FBS) for 5 days. Antibodies against A2B5 cDNA was purified with the cDNA Purification Kit (Enzo Life and GFAP were used to identify type 2 astrocytes. Sciences). The resulting double-stranded DNA was then used to gen- All embryonic rats were obtained from pregnant Wistar rats erate multiple copies of biotinylated cRNA by in vitro transcription bred in the Animal Care Facility at Bengbu Medical College, Bengbu, with the Bioarray HighYield RNA Transcript Labeling Kit (Enzo Life P.R. China. All animal care was carried out in accordance with the Sciences). For each sample, biotinylated cRNA (10 μg) spiked with National Institute of Health Guide for the Care and Use of Laboratory bioB, bioC, bioD, and cre (Hybridization Control) was hybridized